Quantitative analysis of λDNA using SAF-850 One drop accessory

June 11, 2020

Introduction

SAF-850 One Drop accessory is a special accessory, enabling fluorescence measurement by just dropping 5 µL of the sample on a cell and then covering it with another glass. This simple way of measurement is the most suitable and effective for quantitative analysis of multiple samples such as fluorescently-labeled DNA and various kinds of fluorescent dye. In this application data, the measurement result of λDNA labeled with PicoGreen is shown.

Experimental

Measurement system
FP-8200 Spectrofluorometer
SAF-850 One Drop accessory

Measurement procedure

Samples
λDNA labeled with PicoGreen®:  0, 1, 5, 10, 50, 100, 500, 1000 ng/mL

Keywords

One Drop, nucleic acid, reproducibility, linearity

Results

Spectra
The emission spectra of sample with each concentration with Ex. Wavelength of 480 nm are shown in Figure 1. The result indicates that the peak wavelength is 523 nm.

[Measurement parameters]
Measurement mode:  Emission
Excitation wavelength:  485 nm
Measurement range:  510-650 nm
Excitation bandwidth:  10 nm
Emission bandwidth:  10 nm
Scanning speed:  100 nm/min
Data interval:  0.5 nm
Response:  2 sec
Sensitivity:  700V

Figure 1. Emission spectra of λDNA labeled with PicoGreen®

Measurement reproducibility
Table 1 shows the result of 5 times measurements of the sample with each concentration.

[Measurement parameters]
Measurement mode:  Emission
Excitation wavelength:  480 nm
Emission wavelength:  523 nm
Excitation bandwidth:  20 nm
Emission bandwidth:  20 nm
Response:  1 sec
Sensitivity:  620 V

Table 1. Measurement reproducibility

Linearity
The calibration curve was generated using the average value of the results in Table 1. Figure 2 shows the good linearity over a wide concentration range from 1ng/mL to 1000 ng/mL.

Figure 2. Calibration curve of λDNA labeled with PicoGreen®

About the Author

Spectroscopy Group